The Hydroxylamine Oxidizing System of Nitrosomonas Europaea

9723608胡珀NH_2OH被羟胺氧化还原酶氧化(NH_2OH+H_2O-HNO_2+4E-+4H+)，羟胺氧化还原酶是已知最复杂的血红素蛋白，每63 kDa亚基含有7个c-血红素和1个底物结合的血红素p460。后者是一种新的酪氨酸衍生的c-血红素。至少有两对血红素分子以电子方式相互作用，其中一对形成活性部位。第二种羟胺氧化酶“细胞色素P460”有一个金属中心，它是赖氨酸衍生的c-血红素，它与来自微囊藻的细胞色素P-460一起被研究。最近出现了一种HAO的晶体结构，它允许人们提出和测试涉及Fe-N或C-N中间体的不同催化机制，识别和探测活性中心中重要的假体基团和氨基酸，并研究与以前观察到的金属中心之间的磁性相互作用有关的结构元素。本项目的研究重点是HAO和细胞色素P460。方法包括多肽分析、DNA和蛋白质测序、动力学、自杀底物、电位法、光学光谱、EPR光谱、穆斯堡尔光谱、共振拉曼光谱、Endor光谱和X射线结晶学分析。在土壤中，亚硝酸单胞菌有助于将氨氧化成作物所需的氮。在淹水的土壤中，氮肥也可以通过亚硝酸单胞菌转化为气体、一氧化二氮(N20)、一种温室气体和破坏臭氧的催化剂而损失。本文研究的工艺也是废水处理中氨氮去除的限制步骤。最后，亚硝化单胞菌还将催化降解许多氯化污染物。了解所涉及的有机体的生物化学可以更有效地利用化肥-氮，减少一氧化二氮的产生，改善废水处理，并改进受污染土壤的现场生物修复。

9723608 Hooper NH2OH is oxidized (NH2OH + H2O - HNO2 + 4e- + 4H+) by the enzyme hydroxylamine oxidoreductase, "HAO", the most complex hemoprotein known, which contains 7 c-hemes and 1 substrate-binding heme P460 per 63 kDa subunit. The latter is a novel tyrosine-derivatized c-heme. At least two pairs of the hemes interact electronically and one of the pairs forms the active site. A second hydroxylamine-oxidizing enzyme, "cytochrome P460", has one metal center which is a lysine-derivatized c-heme and it, together with a cytochrome P-460 from M.capsulatus, will be studied. A crystal structure for HAO has recently appeared which allows one to propose and test distinct catalytic mechanisms involving Fe-N or C-N intermediates, identify and probe important prosthetic groups and amino acids in the active site, and investigate structural elements responsible for the magnetic interactions previously observed between metal centers. The focus of this project is on HAO and cytochromes P460. Approaches include peptide analysis, DNA and protein sequencing, kinetics, suicide substrates, potentiometry, Optical-, EPR-, Mossbauer-, resonance Raman-, and ENDOR- spectroscopies and X-ray crystallographic analysis. In soils, Nitrosomonas helps oxidize ammonia to the form of nitrogen needed by crops. In flooded soils fertilizer nitrogen can also be lost by conversion by Nitrosomonas to the gas, nitrous oxide (N20), a greenhouse gas and a catalyst of ozone destruction. The process studied here is also the limiting step in the removal of ammonia in waste water treatment. Finally, Nitrosomonas will also catalyze degradation of many chlorinated pollutants. An understanding of the biochemistry of the organism involved could lead to more efficient use of fertilizer-nitrogen, decreased production of nitrous oxide, improvements in the waste water treatment, and improved on-site bioremediation of contaminated soils.